The capacity of genetically modified yeasts to ferment diverse substrates makes them a tool of choice in diverse industrial processes, particularly in the production of ethanol from lignocellulose biomass. Alcoholic fermentation is the process yeasts use in anaerobic media during which sugars are transformed into alcohol. The yeast Saccharomyces cerevisiae, also know as “baker's yeast,” remains the microorganism used most often for alcoholic fermentation.
However, some fermentation inhibitors are found naturally in these substrates and negatively impact ethanol production. This is in particular the case for weak organic acids, particularly for acetic acid, a degradation product of hemicellulose. When yeasts are confronted with the presence of organic acid in their environment, they block their cellular cycle to be able to prepare to react to this new abiotic stress. The fermentation only starts once the cellular resistance machinery is in place. The presence of a weak organic acid accordingly has the consequence of delaying the initiation of the fermentation on glucose, thereby increasing production costs.
The problem related to acetic acid is all the more crucial since it is a very powerful inhibitor for alcoholic fermentation by yeasts and it is found in high concentrations in some fermentation media.
Various means have been described to attempt to counter the effect of fermentation inhibitors, such as for instance detoxifying the fermentation medium, or adapting yeasts to fermentation inhibitors by acclimation or genetic modification. In the case of acetic acid, detoxifying the fermentation medium is a difficult option to implement, particularly industrially. It is therefore necessary to attempt to modify the yeasts themselves.
In this context, the acclimation of yeasts can be achieved by adding the inhibitor into the culture medium, preferably at increasing doses. However, it has been observed that yeast adaptation according to this method is only transient, and disappears quickly when they are again cultivated in a medium devoid of inhibitor. So the method proves to be of little industrial interest, as there phenotypically stable strains are necessary.
In the case of sensitivity to acetic acid, only the genetic modification of yeasts can therefore be envisaged. This can be done either by modification by genetic engineering, targeting specific genes, or classically by crossing strains of interest. Currently, the molecular mechanisms related to the sensitivity or on the contrary the resistance to acetic acid are poorly understood, and insufficient for the methods targeted by genetic engineering.
Accordingly, the method of choice to improve resistance to acetic acid remains yielding yeasts by crossing. However, even though some methods have yielded acetic-acid resistant yeast strains, these are by definition random and cannot guarantee success.
Document WO 2013/178915 describes crossing processes for yeast strains that allow the production of yeasts that can metabolize glucose and are acetic-acid resistant. This method consists in crossing the yeast strain filed at the CNCM under the number I-4538 with the yeast strain filed at the CNCM under the number I-4627, then in selecting a hybrid that can metabolize xylose and, independently, resist acetic acid during the fermentation of glucose.
This hybridization method relies on the capacity of yeasts to reproduce either asexually, or sexually, according to the culture conditions in particular.
Yeast S. cerevisiae is an organism with a haplodiplontic reproductive cycle, i.e. an organism capable of actively multiplying both in the haploid and the polyploid, for example diploid, state.
As long as the medium is favorable, polyploid yeasts are capable of vegetative multiplication by sprouting giving rise to polyploid yeasts. In the case of a medium poor in nitrogen-containing nutrients and containing only a non-fermentable carbon source (for example glycerol, acetate, etc.), the heterozygote cells for the Mat locus enter into meosis and form yeasts having lower ploidy (spores or segregants) by a mechanism called sporulation.
Segregants can multiply by sprouting, giving yeasts having the same genome. Among haploid yeasts two opposing sexual signs are distinguished, called MATa and MATα. Two haploid spores with opposite sexual type can fertilize to yield a diploid yeast.
The haplodiplontic cycle for S. cerevisiae has been widely used to cross sexually compatible segregants (MATa and MATα), particularly in the method called random recombination from mass sporulation and hybridization. In a classic manner, two parental diploid strains (different from the genomic point of view) are used. The sporulation of parental diploid strains is typically induced by cultivating them in conditions where the nitrogen supply is limited and only in the presence of a non-fermentable carbon source. The meiosis operating during this step leads to a genetic cross-fertilization, creating spores with varied genotype. The spores (haploid) obtained for each of the parental strains are then put in contact, to produce diploid (hybrid) strains by fusion. This last step is called the hybridization step.
This method is interesting in that it allows the creation of genetic cross-fertilization from which interesting phenotype traits can emerge. It does however require a step of selecting hybrids on the basis of desired phenotype traits. As an example, in the case of strictly diploid parental strains each presenting a phenotype trait borne by 10 genes, the probability of obtaining the hybrid of interest is estimated at 1/2.097.106. The final selection step is tedious, long and expensive, especially.
Therefore an obvious need exists for improved production methods for acetic-acid resistant yeast strains.